Method for propagating adenoviral vectors encoding inhibitory gene products

ABSTRACT

The invention provides a method of propagating an adenoviral vector. The method comprises (a) providing a cell comprising a cellular genome comprising a nucleic acid sequence encoding a tetracycline operon repressor protein (tetR), and (b) contacting the cell with an adenoviral vector comprising a heterologous nucleic acid sequence encoding a toxic protein. The heterologous nucleic acid sequence is operably linked to a promoter and one or more tetracycline operon operator sequences (tetO), and expression of the heterologous nucleic acid sequence is inhibited in the presence of tetR, such that the adenoviral vector is propagated. The invention also provides a system comprising the aforementioned cell and adenoviral vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of co-pending U.S. applicationSer. No. 14/288,493, filed May 28, 2014, which is a continuation of U.S.application Ser. No. 12/118,008, filed May 9, 2008, now abandoned, whichis a continuation of International Patent Application No.PCT/US2006/060732, filed Nov. 8, 2006, designating the United States,which claims the benefit of U.S. Provisional Patent Application No.60/735,578, filed Nov. 10, 2005.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 2,388 Byte ASCII (Text) file named“725708_ST25.txt” created on Jun. 27, 2016.

BACKGROUND OF THE INVENTION

Delivery of proteins as therapeutics or for inducing an immune responsein biologically relevant amounts has been an obstacle to drug andvaccine development for decades. One solution that has proven to be asuccessful alternative to traditional drug delivery approaches isdelivery of exogenous nucleic acid sequences for production oftherapeutic factors in vivo. Gene transfer vectors ideally enter a widevariety of cell types, have the capacity to accept large nucleic acidsequences, are safe, and can be produced in quantities required fortreating patients. Viral vectors have these advantageous properties andare used in a variety of protocols to treat or prevent biologicaldisorders.

Adenoviral vectors are attractive for gene transfer applications, suchas gene therapy and vaccines as a result of their ability to infect avariety of cell types with high efficiency. Adenoviral vectorscontaining a heterologous transgene under the control of astrongpromoter are potent, achieving expression of the heterologous protein upto 20% of total cell proteins (see, e.g., Massie et al., J. Virol., 72,2289-2296 (1998)). A high level of transgene expression, however, oftenis inhibitory to virus growth, such as when the transgene encodes aprotein that is cytotoxic to a packaging cell. Thus, high expression ofan adenovirus-encoded transgene can prevent the production of viableadenoviral vector particles from naked DNA (see, e.g., Matthews et al.,J. Gen. Virol., 80 (Pt 2), 345-353 (1999)), or reduce the productivityof virus growth within packaging cells (see, e.g., Molin et al., J.Virol., 74, 9002-9009 (2000)).

To better regulate transgene expression within virus packaging cellswhile maintaining vector potency, gene regulation systems have beenemployed in the construction of adenoviral vectors. These systemstypically incorporate transcriptional regulatory proteins into theadenoviral vector or in the target cell (see, e.g., Massie et al.,supra, Goukassian et al., FASEB J., 15, 1877-1885 (2001), Mizuguchi etal., Biochim. Biophys. Acta, 1568, 21-29 (2001), Rubinchik et al., Mol.Ther., 4, 416-426 (2001), Molin et al., J. Virol., 72, 8358-8361 (1998),Hu et al., Cancer Res., 57, 3339-3343 (1997), Edholm et al., J. Virol.,75, 9579-9584 (2001), and U.S. Pat. No. 6,391,612). Such gene regulationsystems, however, often require the use of inducer compounds (e.g.,tetracycline analogs), which increases the time required to generateviable adenoviral vector particles, thereby complicating theirwidespread use.

Therefore, there remains a need for more efficient methods ofpropagating adenoviral vectors encoding transgenes whose expressioninhibits viral growth in host cells. The invention provides such amethod. These and other advantages of the invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of propagating an adenoviral vector,which method comprises (a) providing a cell comprising a cellular genomecomprising a nucleic acid sequence encoding a tetracycline operonrepressor protein (tetR), and (b) contacting the cell with an adenoviralvector having an adenoviral genome comprising a heterologous nucleicacid sequence encoding a protein that is toxic to the cell, wherein theheterologous nucleic acid sequence is operably linked to a promoter andone or more tetracycline operon operator sequences (tetO), so as totransfect the cell with the adenoviral vector. The nucleic acid sequenceencoding tetR is expressed to produce tetR, expression of theheterologous nucleic acid sequence is inhibited in the presence of tetR,and the adenoviral vector is propagated.

The invention also provides a system comprising (a) a cell comprising acellular genome comprising a nucleic acid sequence encoding atetracycline operon repressor protein (tetR), which can be expressed toproduce tetR, and (b) an adenoviral vector having an adenoviral genomecomprising a heterologous nucleic acid sequence encoding a protein thatis toxic to the cell. The heterologous nucleic acid sequence is operablylinked to a promoter and one or more tetracycline operon operatorsequences (tetO), and the adenoviral vector can transfect the cell andbe propagated in the cell.

DETAILED DESCRIPTION OF THE INVENTION

The invention is predicated, at least in part, on the discovery thatadenoviral vectors encoding inhibitory gene products can be producedusing a tetracycline operon-based gene regulation system in whichregulation of gene expression is mediated through the packaging cellline, wherein the addition of inducer compounds is not required.

The invention provides a method of propagating an adenoviral vector.Adenovirus from various origins, subtypes, or mixture of subtypes can beused as the source of the viral genome for the adenoviral vector. Whilenon-human adenovirus (e.g., simian, avian, canine, ovine, or bovineadenoviruses) can be used to generate the adenoviral vector, a humanadenovirus preferably is used as the source of the viral genome for theadenoviral vector of the inventive method. Adenovirus can be of varioussubgroups or serotypes. For instance, an adenovirus can be of subgroup A(e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11,14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32,33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g.,serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and51), or any other adenoviral serotype. Adenoviral serotypes 1 through 51are available from the American Type Culture Collection (ATCC, Manassas,Va.). Preferably, in the context of the inventive method, the adenoviralvector is of human subgroup C, especially serotype 2 or even moredesirably serotype 5. However, non-group C adenoviruses can be used toprepare adenoviral gene transfer vectors for delivery of gene productsto host cells. Preferred adenoviruses used in the construction ofnon-group C adenoviral gene transfer vectors include Ad12 (group A), Ad7and Ad35 (group B), Ad30 and Ad36 (group D), Ad4 (group E), and Ad41(group F). Non-group C adenoviral vectors, methods of producingnon-group C adenoviral vectors, and methods of using non-group Cadenoviral vectors are disclosed in, for example, U.S. Pat. Nos.5,801,030, 5,837,511, and 5,849,561 and International PatentApplications WO 97/12986 and WO 98/53087.

The adenoviral vector can comprise a mixture of subtypes and thereby bea “chimeric” adenoviral vector. A chimeric adenoviral vector cancomprise an adenoviral genome that is derived from two or more (e.g., 2,3, 4, etc.) different adenovirus serotypes. In the context of theinvention, a chimeric adenoviral vector can comprise approximately equalamounts of the genome of each of the two or more different adenovirusserotypes. When the chimeric adenoviral vector genome is comprised ofthe genomes of two different adenovirus serotypes, the chimericadenoviral vector genome preferably comprises no more than about 70%(e.g., no more than about 65%, about 50%, or about 40%) of the genome ofone of the adenovirus serotypes, with the remainder of the chimericadenovirus genome being derived from the genome of the other adenovirusserotype. In one embodiment, the chimeric adenoviral vector can containan adenoviral genome comprising a portion of a serotype 2 genome and aportion of a serotype 5 genome. For example, nucleotides 1-456 of suchan adenoviral vector can be derived from a serotype 2 genome, while theremainder of the adenoviral genome can be derived from a serotype 5genome.

The adenoviral vector of the invention can be replication-competent. Forexample, the adenoviral vector can have a mutation (e.g., a deletion, aninsertion, or a substitution) in the adenoviral genome that does notinhibit viral replication in host cells. The inventive adenoviral vectoralso can be conditionally replication-competent. Preferably, however,the adenoviral vector is replication-deficient in host cells.

By “replication-deficient” is meant that the adenoviral vector requirescomplementation of one or more regions of the adenoviral genome that arerequired for replication, as a result of, for example a deficiency in atleast one replication-essential gene function (i.e., such that theadenoviral vector does not replicate in typical host cells, especiallythose in a human patient that could be infected by the adenoviral vectorin the course of the inventive method). A deficiency in a gene, genefunction, gene, or genomic region, as used herein, is defined as adeletion of sufficient genetic material of the viral genome toobliterate or impair the function of the gene (e.g., such that thefunction of the gene product is reduced by at least about 2-fold,5-fold, 10-fold, 20-fold, 30-fold, or 50-fold) whose nucleic acidsequence was deleted in whole or in part. Deletion of an entire generegion often is not required for disruption of a replication-essentialgene function. However, for the purpose of providing sufficient space inthe adenoviral genome for one or more transgenes, removal of a majorityof a gene region may be desirable. While deletion of genetic material ispreferred, mutation of genetic material by addition or substitution alsois appropriate for disrupting gene function. Replication-essential genefunctions are those gene functions that are required for replication(e.g., propagation) and are encoded by, for example, the adenoviralearly regions (e.g., the E1, E2, and E4 regions), late regions (e.g.,the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2gene), and virus-associated RNAs (e.g., VA-RNA1 and/or VA-RNA-2).

The replication-deficient adenoviral vector desirably requirescomplementation of at least one replication-essential gene function ofone or more regions of the adenoviral genome. Preferably, the adenoviralvector requires complementation of at least one gene function of the E1Aregion, the E1B region, or the E4 region of the adenoviral genomerequired for viral replication (denoted an E1-deficient or E4-deficientadenoviral vector). In addition to a deficiency in the E1 region, therecombinant adenovirus also can have a mutation in the major latepromoter (MLP), as discussed in International Patent ApplicationPublication WO 00/00628. Most preferably, the adenoviral vector isdeficient in at least one replication-essential gene function (desirablyall replication-essential gene functions) of the E1 region and at leastone gene function of the nonessential E3 region (e.g., an Xba I deletionof the E3 region) (denoted an E1/E3-deficient adenoviral vector). Withrespect to the E1 region, the adenoviral vector can be deficient in partor all of the E1A region and/or part or all of the E1B region, e.g., inat least one replication-essential gene function of each of the E1A andE1B regions, thus requiring complementation of the E1A region and theE1B region of the adenoviral genome for replication. The adenoviralvector also can require complementation of the E4 region of theadenoviral genome for replication, such as through a deficiency in oneor more replication-essential gene functions of the E4 region.

When the adenoviral vector is E1-deficient, the adenoviral vector genomecan comprise a deletion beginning at any nucleotide between nucleotides335 to 375 (e.g., nucleotide 356) and ending at any nucleotide betweennucleotides 3,310 to 3,350 (e.g., nucleotide 3,329) or even ending atany nucleotide between 3,490 and 3,530 (e.g., nucleotide 3,510) (basedon the adenovirus serotype 5 genome).

When E2A-deficient, the adenoviral vector genome can comprise a deletionbeginning at any nucleotide between nucleotides 22,425 to 22,465 (e.g.,nucleotide 22,443) and ending at any nucleotide between nucleotides24,010 to 24,050 (e.g., nucleotide 24,032) (based on the adenovirusserotype 5 genome). When E3-deficient, the adenoviral vector genome cancomprise a deletion beginning at any nucleotide between nucleotides28,575 to 29,615 (e.g., nucleotide 28,593) and ending at any nucleotidebetween nucleotides 30,450 to 30,490 (e.g., nucleotide 30,470) (based onthe adenovirus serotype 5 genome).

When the adenoviral vector is deficient in at least onereplication-essential gene function in one region of the adenoviralgenome (e.g., an E1- or E1/E3-deficient adenoviral vector), theadenoviral vector is referred to as “singly replication-deficient.” Aparticularly preferred singly replication-deficient adenoviral vectoris, for example, a replication-deficient adenoviral vector requiring, atmost, complementation of the E1 region of the adenoviral genome, so asto propagate the adenoviral vector (e.g., to form adenoviral vectorparticles).

The adenoviral vector of the invention can be “multiplyreplication-deficient,” meaning that the adenoviral vector is deficientin one or more replication-essential gene functions in each of two ormore regions of the adenoviral genome, and requires complementation ofthose functions for replication. For example, the aforementionedE1-deficient or E1/E3-deficient adenoviral vector can be furtherdeficient in at least one replication-essential gene function of the E4region (denoted an E1/E4- or E1/E3/E4-deficient adenoviral vector),and/or the E2 region (denoted an E1/E2- or E1/E2/E3-deficient adenoviralvector), preferably the E2A region (denoted an E1/E2A- orE1/E2A/E3-deficient adenoviral vector). An adenoviral vector deleted ofthe entire E4 region can elicit a lower host immune response. WhenE4-deficient, the adenoviral vector genome can comprise a deletionbeginning at, for example, any nucleotide between nucleotides 32,805 to32,845 (e.g., nucleotide 32,826) and ending at, for example, anynucleotide between nucleotides 35,540 to 35,580 (e.g., nucleotide35,561) (based on the adenovirus serotype 5 genome), optionally inaddition to deletions in the E1 region (e.g., nucleotides 356 to 3,329or nucleotides 356 to 3,510) (based on the adenovirus serotype 5 genome)and/or deletions in the E3 region (e.g., nucleotides 28,594 to 30,469 ornucleotides 28,593 to 30,470) (based on the adenovirus serotype 5genome). The endpoints defining the deleted nucleotide portions can bedifficult to precisely determine and typically will not significantlyaffect the nature of the adenoviral vector, i.e., each of theaforementioned nucleotide numbers can be +/−1, 2, 3, 4, 5, or even 10 or20 nucleotides.

If the adenoviral vector of the invention is deficient in areplication-essential gene function of the E2A region, the vectorpreferably does not comprise a complete deletion of the E2A region,which deletion preferably is less than about 230 base pairs in length.Generally, the E2A region of the adenovirus codes for a DBP (DNA bindingprotein), a polypeptide required for DNA replication. DBP is composed of473 to 529 amino acids depending on the viral serotype. It is believedthat DBP is an asymmetric protein that exists as a prolate ellipsoidconsisting of a globular Ct with an extended Nt domain. Studies indicatethat the Ct domain is responsible for DBP's ability to bind to nucleicacids, bind to zinc, and function in DNA synthesis at the level of DNAchain elongation. However, the Nt domain is believed to function in lategene expression at both transcriptional and post-transcriptional levels,is responsible for efficient nuclear localization of the protein, andalso may be involved in enhancement of its own expression. Deletions inthe Nt domain between amino acids 2 to 38 have indicated that thisregion is important for DBP function (Brough et al., Virology, 196,269-281 (1993)). While deletions in the E2A region coding for the Ctregion of the DBP have no effect on viral replication, deletions in theE2A region which code for amino acids 2 to 38 of the Nt domain of theDBP impair viral replication. It is preferable that any multiplyreplication-deficient adenoviral vector contains this portion of the E2Aregion of the adenoviral genome. In particular, for example, the desiredportion of the E2A region to be retained is that portion of the E2Aregion of the adenoviral genome which is defined by the 5′ end of theE2A region, specifically positions Ad5(23816) to Ad5(24032) of the E2Aregion of the adenoviral genome of serotype Ad5. This portion of theadenoviral genome desirably is included in the adenoviral vector becauseit is not complemented in current E2A cell lines so as to provide thedesired level of viral propagation.

While the above-described deletions are described with respect to anadenovirus serotype 5 genome, one of ordinary skill in the art candetermine the nucleotide coordinates of the same regions of otheradenovirus serotypes, such as an adenovirus serotype 2 genome, withoutundue experimentation, based on the similarity between the genomes ofvarious adenovirus serotypes, particularly adenovirus serotypes 2 and 5.

In one embodiment of the inventive method, the adenoviral vector cancomprise an adenoviral genome deficient in one or morereplication-essential gene functions of each of the E1 and E4 regions(i.e., the adenoviral vector is an E1/E4-deficient adenoviral vector),preferably with the entire coding region of the E4 region having beendeleted from the adenoviral genome. In other words, all the open readingframes (ORFs) of the E4 region have been removed. Most preferably, theadenoviral vector is rendered replication-deficient by deletion of allof the E1 region and by deletion of a portion of the E4 region. The E4region of the adenoviral vector can retain the native E4 promoter,polyadenylation sequence, and/or the right-side inverted terminal repeat(ITR).

It should be appreciated that the deletion of different regions of theadenoviral vector can alter the immune response of the mammal. Inparticular, deletion of different regions can reduce the inflammatoryresponse generated by the adenoviral vector. Furthermore, the adenoviralvector's coat protein can be modified so as to decrease the adenoviralvector's ability or inability to be recognized by a neutralizingantibody directed against the wild-type coat protein, as described inInternational Patent Application WO 98/40509. Such modifications areuseful for long-term treatment of persistent ocular disorders.

The adenoviral vector, when multiply replication-deficient, especiallyin replication-essential gene functions of the E1 and E4 regions, caninclude a spacer sequence to provide viral growth in a complementingcell line similar to that achieved by singly replication-deficientadenoviral vectors, particularly an E1-deficient adenoviral vector. In apreferred E4-deficient adenoviral vector of the invention wherein the L5fiber region is retained, the spacer is desirably located between the L5fiber region and the right-side ITR. More preferably in such anadenoviral vector, the E4 polyadenylation sequence alone or, mostpreferably, in combination with another sequence exists between the L5fiber region and the right-side ITR, so as to sufficiently separate theretained L5 fiber region from the right-side ITR, such that viralproduction of such a vector approaches that of a singlyreplication-deficient adenoviral vector, particularly a singlyreplication-deficient E1 deficient adenoviral vector.

The spacer sequence can contain any nucleotide sequence or sequenceswhich are of a desired length, such as sequences at least about 15 basepairs (e.g., between about 15 base pairs and about 12,000 base pairs),preferably about 100 base pairs to about 10,000 base pairs, morepreferably about 500 base pairs to about 8,000 base pairs, even morepreferably about 1,500 base pairs to about 6,000 base pairs, and mostpreferably about 2,000 to about 3,000 base pairs in length. The spacersequence can be coding or non-coding and native or non-native withrespect to the adenoviral genome, but does not restore thereplication-essential function to the deficient region. The spacer canalso contain a promoter-variable expression cassette. More preferably,the spacer comprises an additional polyadenylation sequence and/or apassenger gene. Preferably, in the case of a spacer inserted into aregion deficient for E4, both the E4 polyadenylation sequence and the E4promoter of the adenoviral genome or any other (cellular or viral)promoter remain in the vector. The spacer is located between the E4polyadenylation site and the E4 promoter, or, if the E4 promoter is notpresent in the vector, the spacer is proximal to the right-side ITR. Thespacer can comprise any suitable polyadenylation sequence. Examples ofsuitable polyadenylation sequences include synthetic optimizedsequences, BGH (Bovine Growth Hormone), polyoma virus, TK (ThymidineKinase), EBV (Epstein Barr Virus) and the papillomaviruses, includinghuman papillomaviruses and BPV (Bovine Papilloma Virus). Preferably,particularly in the E4 deficient region, the spacer includes an SV40polyadenylation sequence. The SV40 polyadenylation sequence allows forhigher virus production levels of multiply replication deficientadenoviral vectors. In the absence of a spacer, production of fiberprotein and/or viral growth of the multiply replication-deficientadenoviral vector is reduced by comparison to that of a singlyreplication-deficient adenoviral vector. However, inclusion of thespacer in at least one of the deficient adenoviral regions, preferablythe E4 region, can counteract this decrease in fiber protein productionand viral growth. Ideally, the spacer is composed of the glucuronidasegene. The use of a spacer in an adenoviral vector is further describedin, for example, U.S. Pat. No. 5,851,806 and International PatentApplication WO 97/21826.

It has been observed that an at least E4-deficient adenoviral vectorexpresses a transgene at high levels for a limited amount of time invivo and that persistence of expression of a transgene in an at leastE4-deficient adenoviral vector can be modulated through the action of atrans-acting factor, such as HSV ICPO, Ad pTP, CMV-IE86, HIV tat,HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205osteosarcoma cell line that functions like HSV ICPO, or the cellularfactor in PC12 cells that is induced by nerve growth factor, amongothers, as described in for example, U.S. Pat. Nos. 6,225,113,6,649,373, and 6,660,521, and International Patent ApplicationPublication WO 00/34496. In view of the above, a multiply deficientadenoviral vector (e.g., the at least E4-deficient adenoviral vector) ora second expression vector can comprise a nucleic acid sequence encodinga trans-acting factor that modulates the persistence of expression ofthe nucleic acid sequence. Persistent expression of antigenic DNA can bedesired when generating immune tolerance.

Desirably, the adenoviral vector requires, at most, complementation ofreplication-essential gene functions of the E1, E2A, and/or E4 regionsof the adenoviral genome for replication (i.e., propagation). However,the adenoviral genome can be modified to disrupt one or morereplication-essential gene functions as desired by the practitioner, solong as the adenoviral vector remains deficient and can be propagatedusing, for example, complementing cells and/or exogenous DNA (e.g.,helper adenovirus) encoding the disrupted replication-essential genefunctions. In this respect, the adenoviral vector can be deficient inreplication-essential gene functions of only the early regions of theadenoviral genome, only the late regions of the adenoviral genome, andboth the early and late regions of the adenoviral genome. Suitablereplication-deficient adenoviral vectors, including singly and multiplyreplication-deficient adenoviral vectors, are disclosed in U.S. Pat.Nos. 5,837,511, 5,851,806, 5,994,106, 6,127,175, and 6,482,616; U.S.Patent Application Publications 2001/0043922 A1, 2002/0004040 A1,2002/0031831 A1, 2002/0110545 A1, and 2004/0161848 A1; and InternationalPatent Application Publications WO 94/28152, WO 95/02697, WO 95/16772,WO 95/34671, WO 96/22378, WO 97/12986, WO 97/21826, and WO 03/022311.

By removing all or part of, for example, the E1, E3, and E4 regions ofthe adenoviral genome, the resulting adenoviral vector is able to acceptinserts of exogenous nucleic acid sequences while retaining the abilityto be packaged into adenoviral capsids. The nucleic acid sequence can bepositioned in the E1 region, the E3 region, or the E4 region of theadenoviral genome. Indeed, the nucleic acid sequence can be insertedanywhere in the adenoviral genome so long as the position does notprevent expression of the nucleic acid sequence or interfere withpackaging of the adenoviral vector.

If the adenoviral vector is not replication-deficient, ideally theadenoviral vector is manipulated to limit replication of the vector towithin a target tissue. The adenoviral vector can be aconditionally-replicating adenoviral vector, which is engineered toreplicate under conditions pre-determined by the practitioner. Forexample, replication-essential gene functions, e.g., gene functionsencoded by the adenoviral early regions, can be operably linked to aninducible, repressible, or tissue-specific transcription controlsequence, e.g., promoter. In this embodiment, replication requires thepresence or absence of specific factors that interact with thetranscription control sequence. In autoimmune disease treatment, it canbe advantageous to control adenoviral vector replication in, forinstance, lymph nodes, to obtain continual antigen production andcontrol immune cell production. Conditionally-replicating adenoviralvectors are described further in U.S. Pat. No. 5,998,205.

In addition to modification (e.g., deletion, mutation, or replacement)of adenoviral sequences encoding replication-essential gene functions,the adenoviral genome can contain benign or non-lethal modifications,i.e., modifications which do not render the adenovirusreplication-deficient, or, desirably, do not adversely affect viralfunctioning and/or production of viral proteins, even if suchmodifications are in regions of the adenoviral genome that otherwisecontain replication-essential gene functions. Such modificationscommonly result from DNA manipulation or serve to facilitate expressionvector construction. For example, it can be advantageous to remove orintroduce restriction enzyme sites in the adenoviral genome. Such benignmutations often have no detectable adverse effect on viral functioning.For example, the adenoviral vector can comprise a deletion ofnucleotides 10,594 and 10,595 (based on the adenoviral serotype 5genome), which are associated with VA-RNA-1 transcription, but thedeletion of which does not prohibit production of VA-RNA-1.

Similarly, the coat protein of a viral vector, preferably an adenoviralvector, can be manipulated to alter the binding specificity orrecognition of a virus for a viral receptor on a potential host cell.For adenovirus, such manipulations can include deletion of regions ofthe fiber, penton, or hexon, insertions of various native or non-nativeligands into portions of the coat protein, and the like. Manipulation ofthe coat protein can broaden the range of cells infected by a viralvector or enable targeting of a viral vector to a specific cell type.

For example, in one embodiment, the adenoviral vector comprises achimeric coat protein (e.g., a fiber, hexon pIX, pIIIa, or pentonprotein), which differs from the wild-type (i.e., native) coat proteinby the introduction of a nonnative amino acid sequence, preferably at ornear the carboxyl terminus. Preferably, the nonnative amino acidsequence is inserted into or in place of an internal coat proteinsequence. One of ordinary skill in the art will understand that thenonnative amino acid sequence can be inserted within the internal coatprotein sequence or at the end of the internal coat protein sequence.The resultant chimeric viral coat protein is able to direct entry intocells of the adenoviral, vector comprising the coat protein that is moreefficient than entry into cells of a vector that is identical except forcomprising a wild-type adenoviral coat protein rather than the chimericadenoviral coat protein. Preferably, the chimeric adenovirus coatprotein binds a novel endogenous binding site present on the cellsurface that is not recognized, or is poorly recognized, by a vectorcomprising a wild-type coat protein. One direct result of this increasedefficiency of entry is that the adenovirus can bind to and enternumerous cell types which an adenovirus comprising wild-type coatprotein typically cannot enter or can enter with only a low efficiency.

In another embodiment of the invention, the adenoviral vector comprisesa chimeric virus coat protein not selective for a specific type ofeukaryotic cell. The chimeric coat protein differs from the wild-typecoat protein by an insertion of a nonnative amino acid sequence into orin place of an internal coat protein sequence. In this embodiment, thechimeric adenovirus coat protein efficiently binds to a broader range ofeukaryotic cells than a wild-type adenovirus coat, such as described inInternational Patent Application WO 97/20051.

Specificity of binding of an adenovirus to a given cell can also beadjusted by use of an adenovirus comprising a short-shafted adenoviralfiber gene, as discussed in U.S. Pat. No. 5,962,311. Use of anadenovirus comprising a short-shafted adenoviral fiber gene reduces thelevel or efficiency of adenoviral fiber binding to its cell-surfacereceptor and increases adenoviral penton base binding to itscell-surface receptor, thereby increasing the specificity of binding ofthe adenovirus to a given cell. Alternatively, use of an adenoviruscomprising a short-shafted fiber enables targeting of the adenovirus toa desired cell-surface receptor by the introduction of a nonnative aminoacid sequence either into the penton base or the fiber knob.

Of course, the ability of an adenoviral vector to recognize a potentialhost cell can be modulated without genetic manipulation of the coatprotein. For instance, complexing an adenovirus with a bispecificmolecule comprising a penton base-binding domain and a domain thatselectively binds a particular cell surface binding site enables one ofordinary skill in the art to target the vector to a particular celltype.

Suitable modifications to an adenoviral vector are described in U.S.Pat. No. 5,543,328, 5,559,099, 5,712,136, 5,731,190, 5,756,086,5,770,442, 5,846,782, 5,871,727, 5,885,808, 5,922,315, 5,962,311,5,965,541, 6,057,155, 6,127,525, 6,153,435, 6,329,190, 6,455,314,6,465,253, 6,576,456, 6,649,407, 6,740,525; 6,951,755; U.S. PatentApplication Publications 2001/0047081 A1, 2002/0013286 A1, 2002/0151027A1, 2003/0022355 A1, 2003/0099619 A1, 2003/0166286 A1, and 2004/0161848A1; and International Patent Applications WO 95/02697, WO 95/16772, WO95/34671, WO 96/07734, WO 96/22378, WO 96/26281, WO 97/20051, WO98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO 00/15823, WO01/58940, and WO 01/92549. Similarly, it will be appreciated thatnumerous adenoviral vectors are available commercially. Construction ofadenoviral vectors is well understood in the art. Adenoviral vectors canbe constructed and/or purified using methods known in the art (e.g.,using complementing cell lines, such as the 293 cell line, Per.C6 cellline, or 293-ORF6 cell line) and methods set forth, for example, in U.S.Pat. Nos. 5,965,358, 5,994,128, 6,033,908, 6,168,941, 6,329,200,6,383,795, 6,440,728, 6,447,995, 6,475,757, 6,908,762, and 6,913,927;U.S. Patent Application Publications 2002/0034735 A1 and 2004/0063203A1; and International Patent Applications WO 98/53087, WO 98/56937, WO99/15686, WO 99/54441, WO 00/12765, WO 01/77304, and WO 02/29388, aswell as the other references identified herein.

The adenoviral vector of the inventive method comprises an adenoviralgenome comprising a heterologous nucleic acid sequence. A “heterologousnucleic acid sequence” is any nucleic acid sequence that is not obtainedfrom, derived from, or based upon a naturally occurring nucleic acidsequence of the adenoviral vector. By “naturally occurring” is meantthat the nucleic acid sequence can be found in nature and has not beensynthetically modified. The heterologous nucleic acid sequence also isnot obtained from, derived from, or based upon an adenoviral nucleicacid sequence. For example, the heterologous nucleic acid sequence canbe a viral, bacterial, plant, or animal nucleic acid sequence. Asequence is “obtained” from a source when it is isolated from thatsource. A sequence is “derived” from a source when it is isolated from asource but modified in any suitable manner (e.g., by deletion,substitution (mutation), insertion, or other modification to thesequence) so as not to disrupt the normal function of the source gene. Asequence is “based upon” a source when the sequence is a sequence morethan about 70% homologous (preferably more than about 80% homologous,more preferably more than about 90% homologous, and most preferably morethan about 95% homologous) to the source but obtained through syntheticprocedures (e.g., polynucleotide synthesis, directed evolution, etc.).Determining the degree of homology, including the possibility for gaps,can be accomplished using any suitable method (e.g., BLASTnr, providedby GenBank). Notwithstanding the foregoing, the nucleic acid sequencethat makes up the heterologous nucleic acid sequence can be naturallyfound in the adenoviral vector, but located at a nonnative positionwithin the adenoviral genome and/or operably linked to a nonnativepromoter.

The adenoviral vector comprises at least one heterologous nucleic acidsequence as described herein, i.e., the adenoviral vector can compriseone heterologous nucleic acid sequence as described herein or more thanone heterologous nucleic acid sequence as described herein (i.e., two ormore of the heterologous nucleic acid sequences). The heterologousnucleic acid sequence preferably encodes a protein (i.e., one or morenucleic acid sequences encoding one or more proteins). An ordinarilyskilled artisan will appreciate that any type of nucleic acid sequence(e.g., DNA, RNA, and cDNA) that can be inserted into an adenoviralvector can be used in connection with the invention.

In the context of the invention, the heterologous nucleic acid sequencecan encode any suitable protein, but preferably encodes a protein thatis toxic to the cell. Desirably, the protein is a bacterial protein, aviral protein, a plant protein, a parasite protein, a fungi protein, ananimal protein, or an antibiotic. When the heterologous nucleic acidsequence encodes a bacterial protein, the protein can be isolated orderived from any suitable bacterium, including, but not limited toActinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Caulobacter,Chlamydia, Chlorobium, Chromatium, Clostridium, Cytophaga, Deinococcus,Escherichia, Halobacterium, Heliobacter, Hyphomicrobium,Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus,Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema. Desirably, the heterologousnucleic acid sequence encodes a toxin protein isolated or derived fromBacillus anthracis (e.g., protective antigen, lethal factor, or edemafactor), Bordetella pertussis (e.g., adenylate cyclase toxin orpertussis toxin), Vibrio cholerae (e.g., cholera enterotoxin),Escherichia coli (e.g., ST toxin or LT toxin), Shigella dysenteriae(e.g., shiga toxin), Clostridium perfringens (e.g., perfringensenterotoxin), Clostridium botulinum (e.g., botulinum toxin), Clostridiumtetani (e.g., tetanus toxin), Corynebacterium diphtheriae (e.g.,diphtheria toxin), Pseudomonas aeruginosa (e.g., exotoxin A),Staphylococcus aureus (e.g., staphylococcus enterotoxins, toxic shocksyndrome toxin, or exfoliatin toxin), or Streptococcus pyogenes (e.g.,erythrogenic toxin).

The heterologous nucleic acid also can be encode a parasite protein,such as, but not limited to, a parasite of the phylum Sporozoa (alsoreferred to as phylum Apicomplexa), Ciliophora, Rhizopoda, orZoomastigophora. Preferably, the parasite is of the phylum Sporozoa andgenus Plasmodium. The protein can be from any suitable Plasmodiumspecies, but preferably is from a Plasmodium species that infects humansand causes malaria (e.g., Plasmodium falciparum, Plasmodium vivax,Plasmodium ovale, Plasmodium malariae). Suitable Plasmodium proteinsinclude, for example, circumsporozoite protein (CSP), sporozoite surfaceprotein 2 (SSP2), liver-stage antigen 1 (LSA-1), Pf exported protein 1(PfExp-1)/Py hepatocyte erythrocyte protein 17 (PyHEP17), Pf Antigen 2,merozoite surface protein 1 (MSP-1), merozoite surface protein 2(MSP-2), erythrocyte binding antigen 175 (EBA-175), ring-infectederythrocyte surface antigen (RESA), serine repeat antigen (SERA),glycophorin binding protein (GBP-130), histidine rich protein 2 (HRP-2),rhoptry-associated proteins 1 and 2 (RAP-1 and RAP-2), erythrocytemembrane protein 1 (PfEMP1), and apical membrane antigen 1 (AMA-1).

When the heterologous nucleic acid sequence encodes a virus protein, theprotein can be isolated or derived from any suitable virus including,but not limited to, a virus from any of the following viral families:Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus,Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus,Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acuterespiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae,Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virusand Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)),Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2,Dengue virus 3, and Dengue virus 4), Hepadnaviridae (e.g., Hepatitis Bvirus), Herpesviridae (e.g., Human herpesvirus 1, 3, 4, 5, and 6, andCytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae,Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus Aand B), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and humanrespiratory syncytial virus), Parvoviridae, Picornaviridae (e.g.,poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g.,vaccinia virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g.,lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2),Rhabdoviridae, Totiviridae, Crimean-Congo haemorrhagic fever virus,Eastern Equine Encephalitis virus, Hendra virus, Lassa fever virus,Monkeypox virus, Nipah virus, Rift Valley fever virus, South AmericanHaemorrhagic Fever viruses, and Venezuelan Equine Encephalitis virus.

Preferably, at least one protein of the inventive method is a retroviralprotein. The retroviral protein can be, for example, an HIV antigen,such as all or part of the gag, env, or pol proteins, or a fusionprotein comprising any of the gag, env, or pol proteins. Any Glade ofHIV is appropriate for protein selection, including clades A, B, C, MN,and the like. Also preferably, at least one protein encoded by theheterologous nucleic acid sequence is a coronavirus protein, such as aSARS virus protein. Suitable SARS virus proteins for the inventivemethod include, for example, all or part of the E protein, the Mprotein, and the spike protein of the SARS virus. In another embodiment,at least one protein encoded by the heterologous nucleic acid sequenceis an aphthovirus protein, such as a foot-and-mouth disease virus (FMDV)protein. Suitable FMDV proteins include, for example, proteins 1A, 1B,1C, and 1D, collectively referred to as P1, which form the capsidproteins of the virus, proteins 2A, 2B, and 2C (collectively referred toas the P2 protein), and proteins 3A, 3B, 3C, and 3D (collectivelyreferred to as the P3 protein). The FMDV protein also can be an emptyvirus capsid of FMDV. An “empty virus capsid” contains only the portionof the FMDV genome encoding the viral structural proteins and the 3Cprotein, which is required for capsid formation (see Mayr et al.,Virology, 263: 496-506 (1999)), and does not contain infectious viralnucleic acid. Suitable viral proteins also include all or part of Dengueprotein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and DengueD1NS3. The viral peptides specifically recited herein are merelyexemplary as any viral protein can be used in the context of theinvention.

When the heterologous nucleic acid sequence encodes a fungal protein,the protein can be isolated or obtained from any of the followinggenuses: Coccidioides, Candida, Cryptococcus, Trichosporon, Acremonium,Cladophialophora, Pseudallescheria, Rizopus, Scedosporium, Aspergillus,Aureobasidium, Bipolaris, Fusarium, Phialophora, Blastomyces,Histoplasma, or Sporothrix.

When the heterologous nucleic acid sequence encodes a plant protein, theplant protein can be any suitable protein naturally produced by a plant,so long as it is toxic to animal cells (e.g., human cells). Suitableplant toxins include, but are not limited to, lectins (e.g., ricin orabrin), alkaloids, glycosides, oxalates, phenols, resins, volatile oils,and phototoxins (e.g., coumarins).

The heterologous nucleic acid sequence also can encode an animalprotein. In this regard, certain animal proteins are inhibitory toadenovirus replication when such proteins are produced in packagingcells. The heterologous nucleic acid sequence can encode any suitableanimal protein. Examples of suitable animal proteins include, but arenot limited to transforming growth factor β (TGFβ), or nitric oxidesynthase (NOS).

In another embodiment, the heterologous nucleic acid sequence can encodean antibiotic. The antibiotic can be isolated from nature, syntheticallygenerated, isolated from a genetically engineered organism, and thelike. The heterologous nucleic acid sequence can encode any suitableantibiotic. Suitable antibiotics include, but are not limited to,penicillin, ampicillin, cephalosporin, griseofulvin, bacitracin,polymyxin B, amphotericin B, erythromycin, neomycin, streptomycin,tetracycline, vancomycin, gentamicin, rifamycin, and the like.

One of ordinary skill in the art will appreciate that many of theaforementioned proteins, and portions thereof, can be antigenic whenproduced in an animal (e.g., mammalian) cell. An “antigen” is a moleculethat triggers an immune response in a mammal. An “immune response” canentail, for example, antibody production and/or the activation of immuneeffector cells. Thus, the heterologous nucleic acid sequence can encodean antigen which comprises any subunit of any proteinaceous molecule,including a protein or peptide of viral, bacterial, parasitic, fungal,protozoan, prion, cellular, or extracellular origin, which ideallyprovokes an immune response in mammal, preferably leading to protectiveimmunity. The heterologous nucleic acid sequence also can encode a selfantigen, i.e., an autologous protein which the body reacts to as if itis a foreign invader.

Preferably, the nucleic acid is operably linked to (i.e., under thetranscriptional control of) one or more promoter and/or enhancerelements, for example, as part of a promoter-variable expressioncassette. Techniques for operably linking sequences together are wellknown in the art. A “promoter” is a DNA sequence that directs thebinding of RNA polymerase and thereby promotes RNA synthesis. A nucleicacid sequence is “operably linked” to a promoter when the promoter iscapable of directing transcription of that nucleic acid sequence. Apromoter can be native or non-native to the nucleic acid sequence towhich it is operably linked.

Any promoter (i.e., whether isolated from nature or produced byrecombinant DNA or synthetic techniques) can be used in connection withthe invention to provide for transcription of the nucleic acid sequence.The promoter preferably is capable of directing transcription in aeukaryotic (desirably mammalian) cell. The functioning of the promotercan be altered by the presence of one or more enhancers and/or silencerspresent on the vector. “Enhancers” are cis-acting elements of DNA thatstimulate or inhibit transcription of adjacent genes. An enhancer thatinhibits transcription also is termed a “silencer.” Enhancers differfrom DNA-binding sites for sequence-specific DNA binding proteins foundonly in the promoter (which also are termed “promoter elements”) in thatenhancers can function in either orientation, and over distances of upto several kilobase pairs (kb), even from a position downstream of atranscribed region.

Promoter regions can vary in length and sequence and can furtherencompass one or more DNA binding sites for sequence-specific DNAbinding proteins and/or an enhancer or silencer. Enhancers and/orsilencers can similarly be present on a nucleic acid sequence outside ofthe promoter per se. Desirably, a cellular or viral enhancer, such asthe cytomegalovirus (CMV) immediate-early enhancer, is positioned in theproximity of the promoter to enhance promoter activity. In addition,splice acceptor and donor sites can be present on a nucleic acidsequence to enhance transcription.

Any suitable promoter or enhancer sequence can be used in the context ofthe invention. In this respect, the heterologous nucleic acid sequencecan be operably linked to a viral promoter. Suitable viral promotersinclude, for instance, cytomegalovirus (CMV) promoters, such as the CMVimmediate-early promoter (described in, for example, U.S. Pat. Nos.5,168,062 and 5,385,839, and GenBank accession number X17403), promotersderived from human immunodeficiency virus (HIV), such as the HIV longterminal repeat promoter, Rous sarcoma virus (RSV) promoters, such asthe RSV long terminal repeat, mouse mammary tumor virus (MMTV)promoters, HSV promoters, such as the Lap2 promoter or the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci., 78,144-145 (1981)), promoters derived from SV40 or Epstein Barr virus, anadeno-associated viral promoter, such as the p5 promoter, and the like.

Alternatively, the heterologous nucleic acid sequence can be operablylinked to a cellular promoter, i.e., a promoter that drives expressionof a cellular protein. Preferred cellular promoters for use in theinvention will depend on the desired expression profile to produce theantigen(s). In one aspect, the cellular promoter is preferably aconstitutive promoter that works in a variety of cell types, such asimmune cells. Suitable constitutive promoters can drive expression ofgenes encoding transcription factors, housekeeping genes, or structuralgenes common to eukaryotic cells. For example, the Ying Yang 1 (YY1)transcription factor (also referred to as NMP-1, NF-E1, and UCRBP) is aubiquitous nuclear transcription factor that is an intrinsic componentof the nuclear matrix (Guo et al., PNAS, 92, 10526-10530 (1995)). Whilethe promoters described herein are considered as constitutive promoters,it is understood in the art that constitutive promoters can beupregulated. Promoter analysis shows that the elements critical forbasal transcription reside from −277 to +475 of the YY1 gene relative tothe transcription start site from the promoter, and include a TATA andCCAAT box. JEM-1 (also known as HGMW and BLZF-1) also is a ubiquitousnuclear transcription factor identified in normal and tumorous tissues(Tong et al., Leukemia, 12(11), 1733-1740 (1998), and Tong et al.,Genomics, 69(3), 380-390 (2000)). JEM-1 is involved in cellular growthcontrol and maturation, and can be upregulated by retinoic acids.Sequences responsible for maximal activity of the JEM-1 promoter havebeen located at −432 to +101 of the JEM-1 gene relative thetranscription start site of the promoter. Unlike the YY1 promoter, theJEM-1 promoter does not comprise a TATA box. The ubiquitin promoter,specifically UbC, is a strong constitutively active promoter functionalin several species. The UbC promoter is further characterized inMarinovic et al., J. Biol. Chem., 277(19), 16673-16681 (2002).

In the inventive method, the heterologous nucleic acid sequence isoperably linked to a promoter and one or more operator sequences of thetetracycline operon (tetO). The tetracycline operon was originallyidentified in the Tn10 transposon, in which it regulates the expressionof tetracycline resistance genes (see, e.g., Hillen et al. inProtein-Nucleic Acid Interaction, Topics in Molecular and StructuralBiology, Saenger et al., eds., Vol. 10, 143-162, Macmillan, London(1989)). The tetracycline operon, and modified forms thereof, are usedin the art to regulate gene expression in recombinant DNA systems. Inthis regard, the tetracycline regulation system consists of twocomponents: operator sequences (tetO) and a repressor protein (tetR). Inthe absence of tetracycline, the tetR protein is able to bind to thetetO sites and repress transcription of a gene operably linked to thetetO sites. In the presence of tetracycline, however, a conformationalchange in the tetR protein prevents it from binding to the operatorsequences, allowing transcription of operably linked genes to occur. Thetetracycline regulation system has been modified for use in mammaliancells by the generation of a fusion protein combining tetR with thetranscriptional activation domain of the VP16 protein of herpes simplexvirus, which also is referred to as the tet transactivator protein (tTa)(see, e.g., Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89, 5547-5551(1992), and Shockett and Schatz, Proc. Natl. Acad. Sci. USA, 93,5173-5176 (1996)).

The heterologous nucleic acid sequence can be operably linked to anysuitable tetO site and any suitable number of tetO sites, so long asexpression of the heterologous nucleic acid sequence is inhibited in thepresence of tetR. In a preferred embodiment of the invention, theheterologous nucleic acid sequence is operably linked to one or moretetO sites, each of which comprises the nucleotide sequenceAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGA GCT (SEQ ID NO:1). The heterologous nucleic acid sequence preferably is operably linkedto at least one (e.g., 1, 2, 3, 4, 5, 6, or more) tetO sequence, butmore preferably is operably linked to at least two (e.g., 2, 3, 4, 5, 6,or more) tetO sequences.

The one or more tetO sequences can be located in any suitable positionwith respect to the heterologous nucleic acid sequence and the promoter.In this regard, the tetO sequences can be located upstream of both thepromoter and the heterologous nucleic acid sequence. Alternatively, thetetO sequences can be located between the promoter and the heterologousnucleic acid sequence. In another embodiment, the one or more tetOsequences can be located downstream of both the promoter and theheterologous nucleic acid sequence. In addition, the one or more tetOsequences need not be positioned in tandem. For example, one tetOsequence can be located upstream of the promoter, while a second tetOsequence can be located downstream of the promoter and upstream of theheterologous nucleic acid sequence.

Operable linkage of a heterologous nucleic acid sequence to a promoterand tetO sequences is within the skill of the art, and can beaccomplished using routine recombinant DNA techniques, such as thosedescribed in, for example, Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y. (1994).

To optimize protein production, preferably the heterologous nucleic acidsequence further comprises a polyadenylation site following the codingsequence of the heterologous nucleic acid sequence. Any suitablepolyadenylation sequence can be used, including a synthetic optimizedsequence, as well as the polyadenylation sequence of BGH (Bovine GrowthHormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein BarrVirus), and the papillomaviruses, including human papillomaviruses andBPV (Bovine Papilloma Virus). A preferred polyadenylation sequence isthe SV40 (Human Sarcoma Virus-40) polyadenylation sequence. Also,preferably all the proper transcription signals (and translationsignals, where appropriate) are correctly arranged such that the nucleicacid sequence is properly expressed in the cells into which it isintroduced. If desired, the nucleic acid sequence also can incorporatesplice sites (i.e., splice acceptor and splice donor sites) tofacilitate mRNA production.

The construction of adenoviral vectors is well understood in the art.Adenoviral vectors can be constructed and/or purified using the methodsset forth, for example, in U.S. Pat. Nos. 5,965,358, 6,168,941,6,329,200, 6,383,795, 6,440,728, 6,447,995, 6,475,757, 6,573,092, and6,586,226, and U.S. Patent Application Publication Nos. 2003/0170899 A1,2003/0203469 A1, and 2003/0203480 A1, and International PatentApplication Publications WO 98/53087, WO 98/56937, WO 99/15686, WO99/54441, WO 00/12765, WO 01/77304, WO 02/29388, WO 02/31169, and WO03/39459 as well as the other references identified herein. Non-group Cadenoviral vectors, including adenoviral serotype 35 vectors, can beproduced using the methods set forth in, for example, U.S. Pat. Nos.5,837,511 and 5,849,561, and International Patent ApplicationPublications WO 97/12986 and WO 98/53087. Moreover, numerous adenoviralvectors are available commercially.

The inventive method further comprises providing a cell having acellular genome comprising a nucleic acid sequence encoding atetracycline operon repressor protein (tetR). The cell can be anysuitable cell which can propagate adenoviral vectors when infected withsuch vectors or with nucleic acid sequences encoding the adenoviralgenome. In this regard, the cell desirably comprises a genome that canincorporate and preferably retain a nucleic acid encoding an adenoviralgene product that complements in trans for a deficiency in one or moreregions of an adenoviral genome. Most preferably, the cell can propagatea suitable replication-deficient adenoviral vector upon infection withan appropriate replication-deficient adenoviral vector or transfectionwith an appropriate replication-deficient viral genome.

Particularly desirable cell types are those that support high levels ofadenovirus propagation. The cell preferably produces at least about10,000 viral particles per cell and/or at least about 3,000 focusforming units (FFU) per cell. More preferably, the cell produces atleast about 100,000 viral particles per cell and/or at least about 5,000FFU per cell. Most preferably, the cell produces at least about 200,000viral particles per cell and/or at least about 7,000 FFU per cell.

Preferably, the cell is, or is derived from, an anchorage dependentcell, but which has the capacity to grow in suspension cultures. In oneembodiment, the cell can be a primary cell. By “primary cell” is meantthat the cell does not replicate indefinitely in culture. Examples ofsuitable primary cells include, but are not limited to, human embryonickidney (HEK) cells, human retinal cells, and human embryonic retinal(HER) cells. In another embodiment, the cell can be a transformed cell.The cell is “transformed” in that the cell has the ability to replicateindefinitely in culture. Examples of suitable transformed cells includerenal carcinoma cells, CHO cells, KB cells, HEK-293 cells, SW-13 cells,MCF7 cells, HeLa cells, Vero cells, neural cells (e.g., BE(2)-M17 cellsand SK-N-MC cells), and lung carcinoma cells. Complementing cell linesfor producing the adenoviral vector include, but are not limited to, 293cells (described in, e.g., Graham et al., J. Gen. Virol., 36, 59-72(1977)), PER.C6 cells (described in, e.g., International PatentApplication WO 97/00326, and U.S. Pat. Nos. 5,994,128 and 6,033,908),and 293-ORF6 cells (described in, e.g., International Patent ApplicationWO 95/34671 and Brough et al., J. Virol., 71, 9206-9213 (1997)).Additional complementing cells are described in, for example, U.S. Pat.Nos. 6,677,156 and 6,682,929, and International Patent ApplicationPublication WO 03/20879.

The cell comprises a cellular genome comprising a nucleic acid sequenceencoding a tetracycline operon repressor protein (tetR). Like the tetOsequence, the tetR protein was originally identified in the Tn10transposon as part of the tetracycline operon (see Hillen et al.,supra). The tetR protein preferably comprises the amino acid sequence ofSEQ ID NO: 2 (GenBank Accession No. J01830, GI No. 154846).

While it is preferred that the nucleic acid sequence encoding the tetRprotein encodes a wild-type tetR protein (such as is set forth in SEQ IDNO: 2), the nucleic acid sequence alternatively can encode any suitablevariant of the tetR protein. A variant of the tetR protein retains theability to bind to tetO sequences and repress transcription of a nucleicacid sequence operably linked thereto. A variant tetR protein preferablyis produced by introducing one or more mutations (e.g., point mutations,deletions, insertions, etc.) into the nucleic acid sequence encoding awild type tetR protein. Such mutations are introduced in the nucleicacid sequence to effect one or more amino acid substitutions in anencoded tetR protein. Thus, where mutations are introduced in thenucleic acid sequence encoding the tetR protein, such mutationsdesirably will effect a substitution in the encoded tetR protein wherebycodons encoding positively-charged residues (H, K, and R) aresubstituted with codons encoding positively-charged residues, codonsencoding negatively-charged residues (D and E) are substituted withcodons encoding negatively-charged residues, codons encoding neutralpolar residues (C, G, N, Q, S, T, and Y) are substituted with codonsencoding neutral polar residues, and codons encoding neutral non-polarresidues (A, F, I, L, M, P, V, and W) are substituted with codonsencoding neutral non-polar residues. In addition, the nucleic acidsequence can encode a homolog of a tetR protein. A homolog of a tetRprotein, whether wild-type or mutant, can be any peptide, polypeptide,or portion thereof, that is more than about 70% identical (preferablymore than about 80% identical, more preferably more than about 90%identical, and most preferably more than about 95% identical) to thetetR protein at the amino acid level. The degree of amino acid identitycan be determined using any method known in the art, such as the BLASTsequence database. Furthermore, a homolog of the tetR protein can be anypeptide, polypeptide, or portion thereof, which hybridizes to the tetRprotein under at least moderate, preferably high, stringency conditions.Exemplary moderate stringency conditions include overnight incubation at37° C. in a solution comprising 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.,or substantially similar conditions, e.g., the moderately stringentconditions described in Sambrook et al., supra. High stringencyconditions are conditions that use, for example (1) low ionic strengthand high temperature for washing, such as 0.015 M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C., (2) employa denaturing agent during hybridization, such as formamide, for example,50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1%Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer atpH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C.,or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at (i) 42° C. in 0.2×SSC,(ii) 55° C. in 50% formamide, and (iii) 55° C. in 0.1×SSC (preferably incombination with EDTA). Additional details and an explanation ofstringency of hybridization reactions are provided in, e.g., Ausubel etal., supra.

Replication-deficient adenoviral vectors are typically produced incomplementing cell lines that provide gene functions not present in thereplication-deficient adenoviral vectors, but required for viralpropagation, at appropriate levels in order to generate high titers ofviral vector stock. Thus, in addition to the nucleic acid sequenceencoding a tetR protein, the cell preferably comprises, integrated intothe cellular genome, adenoviral nucleic acid sequences which encode genefunctions required for adenoviral propagation. A preferred cellcomplements for at least one and preferably all replication-essentialgene functions not present in a replication-deficient adenovirus. Thecell can complement for a deficiency in at least onereplication-essential gene function encoded by the early regions, lateregions, viral packaging regions, virus-associated RNA regions, orcombinations thereof, including all adenoviral functions (e.g., toenable propagation of adenoviral amplicons). Most preferably, the cellcomplements for a deficiency in at least one replication-essential genefunction (e.g., two or more replication-essential gene functions) of theE1 region of the adenoviral genome, particularly a deficiency in areplication-essential gene function of each of the E1A and E1B regions.In addition, the cell can complement for a deficiency in at least onereplication-essential gene function of the E2 (particularly as concernsthe adenoviral DNA polymerase and terminal protein) and/or E4 regions ofthe adenoviral genome.

Desirably, a cell that complements for a deficiency in the E4 regioncomprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein.Such a cell desirably comprises at least ORF6 and no other ORF of the E4region of the adenoviral genome. The ORF-6 of the E4 region of theadenoviral genome preferably is an ORF-6 of the E4 region of a humanadenoviral genome, such as a serotype 5 or serotype 2 adenoviral genome.In addition, the ORF-6 of the E4 region of the adenoviral genome can beoperably linked to any suitable promoter, but preferably is operablylinked to an inducible promoter. Any suitable inducible promoter may beused to regulate the ORF-6 of the E4 region of the adenoviral genome,and suitable inducible promoters are known in the art. In a preferredembodiment of the invention, the inducible promoter is a sheepmetallothionine promoter.

The cell preferably is further characterized in that it contains thecomplementing genes in a non-overlapping fashion with the adenoviralvector, which minimizes, and practically eliminates, the possibility ofthe vector genome recombining with the cellular DNA. Accordingly, thepresence of replication competent adenoviruses (RCA) is minimized if notavoided in the vector stock, which, therefore, is suitable for certaintherapeutic purposes, especially vaccination purposes. The lack of RCAin the vector stock avoids the replication of the adenoviral vector innon-complementing cells. Construction of such a complementing cellinvolves standard molecular biology and cell culture techniques, such asthose described by Sambrook et al., supra, and Ausubel et al., supra).

In some instances, the cellular genome need not comprise nucleic acidsequences, the gene products of which complement for all of thereplication-essential deficiencies of a replication-deficient adenoviralvector. One or more replication-essential gene functions lacking in areplication-deficient adenoviral vector can be supplied by a helpervirus, e.g., an adenoviral vector that supplies in trans one or moreessential gene functions required for replication of the desiredadenoviral vector. Helper virus is often engineered to prevent packagingof infectious helper virus. For example, one or morereplication-essential gene functions of the E1 region of the adenoviralgenome can be provided by the complementing cell, while one or morereplication-essential gene functions of the E4 region of the adenoviralgenome can be provided by a helper virus.

In accordance with the inventive method, the adenoviral vector contactsthe cell so as to transfect the cell with the adenoviral vector, suchthat (a) the nucleic acid sequence encoding tetR is expressed to producetetR, (b) expression of the heterologous nucleic acid sequence isinhibited in the presence of tetR, and (c) the adenoviral vector ispropagated. The cell can be contacted with the adenoviral vector usingany suitable method known in the art. Preferably, the cell istransfected with the adenoviral vector in vitro using standardtechniques (e.g., calcium phosphate precipitated transfection).

Upon uptake of the adenoviral vector by the cell, the tetR proteinproduced by the cell desirably binds to the one or more tetO sequencesoperably linked to the heterologous nucleic acid sequence of theadenoviral vector. As discussed above, tetR binding to tetO sequencesprevents the transcriptional machinery from accessing the promoteroperably linked to the heterologous nucleic acid sequence, therebyinhibiting expression of the heterologous nucleic acid sequence. Theexpression of the heterologous nucleic acid sequence is “inhibited” whenthe level of expression (typically and preferably transcription) of theheterologous nucleic acid sequence in the presence of tetR is at mostabout 80% (e.g., no more than about 80%, about 70%, or about 60%) thelevel of expression of the heterologous nucleic acid sequence in theabsence of tetR. Preferably, the level of expression of the heterologousnucleic acid sequence in the presence of tetR is at most about 50%(e.g., no more than about 50%, about 40%, or about 30%) the level ofexpression of the heterologous nucleic acid sequence in the absence oftetR. More preferably, the level of expression of the heterologousnucleic acid sequence in the presence of tetR is at most about 20%(e.g., no more than about 20%, about 10%, about or about 5%) the levelof expression of the heterologous nucleic acid sequence in the absenceof tetR. Ideally, expression of the heterologous nucleic acid sequenceis completely inhibited in the presence of tetR.

One of ordinary skill in the art will appreciate that expression of thenucleic acid sequence encoding the tetR protein in the cell increasesthe yield of adenoviral vectors encoding the heterologous nucleic acidsequence per cell when compared to the yield of adenoviral vectors percell when the nucleic acid sequence encoding tetR is not expressed inthe cell. Expression of the tetR protein preferably increases the yieldof adenoviral vector at least about 5-fold, and more preferablyincreases the yield of adenoviral vector at least about 20-fold, ascompared to the yield of adenoviral vectors when the cell does notexpress the tetR protein.

The invention further provides a system comprising a cell comprising acellular genome comprising a nucleic acid sequence encoding atetracycline operon repressor protein (tetR), which can be expressed toproduce tetR, and an adenoviral vector. The adenoviral vector has anadenoviral genome comprising a heterologous nucleic acid sequenceencoding a protein that is toxic to the cell, wherein the heterologousnucleic acid sequence is operably linked to a promoter and one or moretetracycline operon operator sequences (tetO), and wherein theadenoviral vector can transfect the cell and be propagated in the cell.Descriptions of the adenoviral vector, the tetO sequences, the cell, andthe tetR protein set forth above in connection with other embodiments ofthe invention also are applicable to those same aspects of the aforesaidsystem.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of inhibiting gene expression from anadenoviral vector according to the inventive method.

An oligonucleotide containing two copies of the tet operator(5′-AGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGA GCT-3′) (SEQID NO: 1) was self-annealed, digested with SacI, and inserted at theSacI site between the TATA box and transcription start site of the CMVenhancer/promoter (GenBank X17403, nucleotides 174,314 to 173,566). Anartificial untranslated sequence (UTR) of 144 base pairs and 3′ splicesite sequences were inserted downstream of the CMV sequences, followedby a nucleic acid sequence encoding green fluorescent protein (GFP) anda simian virus-40 (SV40) polyadenylation signal. The resulting CMV-tetOexpression cassette was transferred to a shuttle plasmid containingadenovirus type 5 nucleotides 1-355 and 3333-5793 or 3511-5793 flankingthe expression cassette and a restriction site for linearization.

Adenoviral vector genomes were constructed using the AdFast method (seeU.S. Pat. No. 6,329,200). Briefly, E. coli strain BIDE3 was transfectedwith 100 ng of shuttle plasmid containing the CMV-tetO expressioncassette and 100 ng of a GV.11 base plasmid. The desired recombinantplasmids, containing deletions in the E1, E3, and E4 regions of theadenoviral genome and the expression cassette were identified byrestriction digestion of DNA from individual bacterial colonies. Theplasmids were further purified by transformation of recombinationnegative DH5a E. coli and single-colony isolation by standardmicrobiological methods. Isolation of a single genetic clone of thefinal vector genome was achieved by two sequential colony-growth stepsin bacteria. The adenoviral vector plasmid structures were confirmed byrestriction digestion analysis and DNA sequencing.

Cell populations of 293 and 293-ORF6 (293 cells that express the Ad5 34kDa E4 ORF6 protein (Brough et al., J. Virol., 70, 6497-6501 (1996))carrying the episomal plasmid pREPrsv(Koz-tetR)BghpA (293TetR and293-ORF6TetR, respectively) were generated by transfection of 2 μg ofcircular plasmid and addition of hygromycin to 150 μg/ml in the cellculture medium. The transfected cell populations were maintained underhygromycin selection. The expression of tetR protein was confirmed byWestern blot analysis of 293TetR and 293-ORF6TetR extracts, whichdemonstrated that the cell lines that maintained the tetR episome wereexpressing tetR protein. To generate the stable transfectant cell line,293-ORF6NT, 293-ORF6 cells were transfected with 2 μg of HpaI-linearizedpRSVTetR.hyg plasmid. After 24 hours the cells were split to ten 10 cmdishes and incubated in 250 μg/ml hygromycin.

To demonstrate functional repressor activity, 293TetR cells weretransduced with the above-described E1-, E3-, E4-deleted, adenoviralvectors (Rasmussen et al., Cancer Gene Ther., 9, 951-957 (2002))comprising a nucleic acid sequence encoding GFP expressed under thecontrol of either a CMV promoter (Adf.11D) or the CMV-tetO promoter(AdtetO.f.11D). The fluorescence intensity of 293TetR cells transducedwith AdtetO.f.11D was much lower than that of cells transduced byAdf.11D. The addition of 2 μg/ml doxycycline (dox) to cultures of293TetR cells transduced by the AdtetO.f.11D adenoviral vector resultedin fluorescent intensity similar to that of 293TetR cells transduced byAdf.11D.

To determine whether the lower protein levels observed with thetetR/tetO system were due to reduced levels of transcription, therelative steady-state levels of GFP mRNA were determined by Northernblot analysis of cells productively infected with 1 or 10 focus formingunits (FFU) per cell of AdtetO.f.11D. Steady-state GFP mRNA was reducedearly (6 hours post-infection or h.p.i.) and late (24 h.p.i.) in293-ORF6TetR cells compared to 293-ORF6. Thus, the lower level ofprotein products during virus replication was due to repression oftranscription. Moreover, binding of tetR protein to adenovirus DNA didnot affect virus propagation or growth since there was no differencebetween marker gene vectors with and without the tetR/tetO system.

The results of this example demonstrate that gene expression from anadenoviral vector comprising tetO sequences can be inhibited in cellsexpressing a functional tetR protein.

Example 2

This example demonstrates a method of inhibiting gene expression from anadenoviral vector according to the inventive method.

293 cells expressing tetR (293TetR) and lacking tetR (293BB) weregenerated as described in Example 1. Cells were infected with E1-deletedadenoviral vectors that expressed secreted alkaline phosphatase (SEAP)from constitutive (AdSeap) and regulatable (AdTetO.Seap) expressioncassettes. The level of SEAP activity in the culture medium wasdetermined (Phospha-Light™ Kit, Applied Biosystems, Foster City, Calif.)at three early phase time points: 8, 10, and 12 hours post-infection(h.p.i.) and at one time point after significant DNA replication (24h.p.i.). The level of SEAP activity was specifically reduced in the293TetR cells infected with AdtetO.Seap. SEAP activity was not reducedin 293BB cells infected with AdtetO.Seap, as compared to 293BB cellsinfected with AdSeap. The level of SEAP activity was reduced by the tetOvector-tetR cell combination more than 10-fold at the early time points,although the reduction in activity decreased to approximately 3-fold by24 h.p.i. There was no effect of the repressor on SEAP expression fromthe adenoviral vector that did not contain tetO sequences (i.e.,AdSeap+293TetR compared to AdSeap+293BB).

Similar experiments were performed using E1-, E3-, E4-deleted adenoviralvectors. In particular, 293-ORF6 cells and 293-ORF6TetR cells (seeExample 1) were infected with SEAP-encoding adenoviral vectors AdS.11Dand AdTetO.S.11D. AdS.11D and AdTetO.S.11D expressed comparable amountsof SEAP in 293-ORF6 cells. In 293-ORF6TetR cells, the expression of SEAPby AdTetO.S.11D was significantly reduced as compared to SEAP expressionby AdTetO.S.11D in 293-ORF6 cells. The greatest differences in SEAPactivity between AdTetO.S.11D infected 293-ORF6 and 293-ORF6TetR cellsoccurred at the early phase time points of 6 and 8 h.p.i. (>10-fold).SEAP activity was reduced about 7 fold at 12 h.p.i., and about 3-fold at24 h.p.i. The addition of 2 μg/ml doxycycline (dox) to cultures of293-ORF6TetR cells at the time of infection with AdTetO.S.11D resultedin the expression of SEAP.

The results of this example demonstrate that gene expression from anadenoviral vector comprising tetO sequences can be inhibited in cellsexpressing a functional tetR protein early in the virus growth cycle.The results of this example also suggest that inhibition of geneexpression is affected by the number of vector genomes present withinthe cell.

Example 3

This example demonstrates a method of propagating an adenoviral vectorcomprising a nucleic acid sequence encoding a toxic protein inaccordance with the inventive method.

Seven adenoviral vectors comprising an expression cassette under thecontrol of a CMV promoter inserted into a deleted E1 region could not bepropagated to form a stock of viable adenoviral vectors using standard293 or 293-ORF6 cells. The transgenes encoded by the adenoviral vectorsincluded human transforming growth factor beta-1 (TGFβ) (Wettergreen etal., Eur. J. Oral Sci., 109, 415-421 (2001), two peptide antibiotics,two viral envelope glycoproteins, and two malaria parasite proteins. Incontrast, adenoviral vectors encoding each of these genes could bepropagated using the tetR/tetO system described herein. To illustrate,an E1-deleted adenoviral vector comprising the CMV-tetO expressioncassette was constructed in accordance with the description herein (see,e.g., Example 1). The adenoviral vector encoded an activated form ofTGFβ known to have a three to five-fold higher biological activity thanwild-type TGFβ (Brunner et al., J. Biol. Chem., 264, 13660-13664 (1989))(AdtetO.TGFβ). To determine the effect of high level TGFβ expression onadenoviral vector propagation, 293 and 293TetR cells were infected withAdtetO.TGFp. At 12 hours post-infection, approximately 1.7-fold moreTGFβ was detected in the culture medium of 293 cells as compared to293TetR cells. However, by 24 h.p.i., significantly more TGFβ hadaccumulated in the culture medium of 293TetR cells (t-test, p=0.01).Despite the high level of TGFβ protein accumulation, there was a 3-foldhigher yield of infectious viral particles from 293TetR cells at 24hours post-infection.

The results of this example demonstrate that an adenoviral vectorencoding a toxic protein (such as TGFβ) can be propagated in accordancewith the inventive method. The results of this example also suggest thatadenoviral propagation is less refractory to toxic protein, specificallyTGFβ, inhibition late in infection.

Example 4

This example demonstrates a method of propagating an adenoviral vectorcomprising a nucleic acid sequence encoding a toxic protein inaccordance with the inventive method.

An adenoviral vector expressing high levels of a modified HIV-1 envelopegene, gp140 (Yang et al., J. Virol., 78, 4029-4036 (2004)) could not beefficiently propagated. Four attempts to propagate the adenoviral vectorfailed before the fifth attempt was successful. The yield of virusprogeny in the fifth attempt was 10-fold lower than expected.

Three different approaches were attempted to overcome the inhibition tovector generation and growth. First, the gp140 nucleic acid sequence wasaltered to remove potential inhibitory areas of the gp140 envelopeprotein. Deletion of protein coding regions of gp140 to generategp140dV12 (Yang et al., supra) resulted in a high level of gp140 geneexpression. The second approach entailed deleting the introns from theCMV expression cassette (hCMVΔ) to decrease the level of gp140expression. This modification resulted in a 10-fold decrease in gp140expression. While both of these approaches were successful at restoringefficient generation of the adenoviral vectors and improved adenovirusproduction yield, the modifications to the gp140 coding sequence andexpression thereof were significant. In contrast, the third approach,which entailed inhibiting gp140 expression using the tetR/tetO tetsystem discussed above, resulted in efficient viral propagation and highvirus yield without altering the gene product or reducing the potency ofthe adenoviral vector. A summary of the results is set forth in thefollowing table.

Relative Viral Viral Relative Propa- Yield^(a) Expres- gation (# of sionSuccess/ prepa- Adenoviral Vector Cell Line (%) Attempts rations)hCMV.Luciferase 293-ORF6 100 1/1 100% (6) hCMV.gp140 293-ORF6 100 1/5 6% (3) hCMV.gp140dV12 293-ORF6 100 1/1  88% (3) hCMVA.gp140 293-ORF6 10 3/3  84% (5) hCMVtetO.gp140 293-ORF6 100 0/1 n.a. hCMVtetO.gp140293-ORF6TetR Repressed 1/1 100% (4) ^(a)Relative to the hCMV.Luciferaseadenoviral vector

The results of this example demonstrate that an adenoviral vectorencoding a toxic protein (such as gp140) can be propagated in accordancewith the inventive method.

Example 5

This example demonstrates a method of propagating an adenoviral vectorcomprising a nucleic acid sequence encoding a toxic protein inaccordance with the inventive method.

An E1-deleted adenoviral vector encoding human inducible nitric oxidesynthase (iNOS) was grown to high titer only if an iNOS inhibitor wasincluded in the culture medium, demonstrating the inhibitory effect ofiNOS overexpression on adenoviral vector replication. Even with the useof inhibitors, however, the iNOS adenoviral vector was propagated at arelatively low titer (10⁹ particle forming units (PFU)/mL) (Shears etal., J. Am. Coll. Surg., 187, 295-306 (1998)). The preparations ofadenoviral vectors containing a CMV-iNOS expression cassette wererapidly overtaken by replication competent adenovirus (RCA) and mutatedvectors containing deletions of the iNOS expression cassette, implying aselective pressure against the expression of iNOS.

To prevent RCA formation and alleviate the negative effects of iNOSoverexpression, an E1-, E3-, and E4-deleted adenoviral vector containinga CMV-TetO-iNOS expression cassette was constructed in accordance withthe description herein. In addition, a PCR assay was developed to assessthe integrity of the expression cassette. Two AdFAST vector plasmidswith identical CMV-tetO-iNOS expression cassettes were generated. Thetwo adenoviral vectors produced via the AdFAST method differed only inthe fiber protein, expressing either a wild-type fiber(AdtetO.hiNOS.11D) or a fiber containing a seven amino acid C-terminaladdition (AdtetO.hiNOS.F(pK7).11D (Wickham et al., J. Virol., 71,8221-8229 (1997)).

293-ORF6 and 293-ORF6TetR cells were transfected with each vector, andlysates were passaged in parallel until cytopathic effect (c.p.e.) onthe cells was observed. The iNOS adenoviral vectors propagated on293-ORF6TetR cells achieved sufficient titer in two passages to generategreater than 50% c.p.e. of 1×10⁶ cells. Subsequent generation of cesiumchloride purified stocks yielded titers averaging 2.7×10″ FFU/mL with anaverage particle:FFU ratio of 8. Transgene expression and activity ofiNOS was confirmed by quantitation of total nitric oxide in transducedcell supernatants (R & D Systems, Minneapolis, Minn.). In comparison,growth of the vectors on 293-ORF6 cells was much slower. Although theadenoviral genome of AdtetO.iNOS.F(pK7).11D was detected by a PCR assaythroughout the virus passages on 293-ORF6 cells, the vector did notachieve sufficient titer to induce c.p.e. on the cells even after sevenpassages.

Infected cell lysates of equal vector passage number were assayed forrearrangements of the expression cassette by PCR analysis. The expectedfull length amplification product was detected with all adenoviralvectors, and there were no unexpected amplicons detectable in theadenoviral vector preparations performed on 293-ORF6TetR cells. However,production and propagation of the iNOS adenoviral vectors on 293-ORF6cells yielded unexpected amplicons smaller than the full length product(i.e., approximately 2.1 kb in AdtetO.iNOS.11D and approximately 1.1 kbin AdtetO.iNOS.F(pK7).11D). The 2.1 kb and 1.1 kb PCR products werepurified from the agarose gel and sequenced. The 2.1 kb ampliconcontained a 2.9 kb deletion of the 3-prime end of the expressioncassette consisting of 80% of the iNOS ORF and the entire SV40polyadenylation site. Similarly, the 1.1 kb amplicon contained a 3.8 kbdeletion of the 5-prime end of the expression cassette consisting of theCMV promoter, leaving only 232 bases of the CMV enhancer, and the entireiNOS ORF.

In addition, an E1-, E3-deleted CMV-tetO-iNOS adenoviral vector wasconstructed by the AdFAST method, propagated on 293TetR cells (Wang etal., Mol. Ther., 7, 597-603 (2003)), and was demonstrated to be free ofRCA (approximately 10¹⁰ pu tested), and no E1 region deletions weredetected by PCR. Thus, as a result of transcriptional repression, thetetR/tetO system was effective in preventing the overgrowth of culturesby adenovectors with non-functional expression cassettes.

The results of this example demonstrate that an adenoviral vectorencoding a toxic protein (such as iNOS) can be propagated in accordancewith the inventive method.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of propagating a non-subgroup C adenoviral vector, whichmethod comprises: (a) providing a cell comprising a cellular genomecomprising a nucleic acid sequence encoding a tetracycline operonrepressor protein (tetR), (b) expressing the nucleic acid sequenceencoding tetR to produce tetR, and (c) contacting the cell with anon-subgroup C adenoviral vector in the absence of tetracycline, whereinthe non-subgroup C adenoviral vector has an adenoviral genome comprisinga heterologous nucleic acid sequence, wherein the heterologous nucleicacid sequence (i) encodes a protein that inhibits propagation of theadenoviral vector in the cell and (ii) is operably linked to a promoterand one or more tetracycline operon operator sequences (tetO), so as totransfect the cell with the non-subgroup C adenoviral vector, whereinexpression of the heterologous nucleic acid sequence is inhibited in thepresence of tetR, and a non-subgroup C adenoviral vector is propagated.2. The method of claim 1, wherein the non-subgroup C adenoviral vectoris replication-deficient.
 3. The method of claim 2, wherein thenon-subgroup C adenoviral vector requires, at most, complementation ofthe E1 region of the adenoviral genome for replication.
 4. The method ofclaim 2, wherein the non-subgroup C adenoviral vector requires, at most,complementation of the E4 region of the adenoviral genome forreplication.
 5. The method of claim 2, wherein the non-subgroup Cadenoviral vector has an adenoviral genome devoid of all of the E1region and at least a portion of the E4 region, and the adenoviralvector requires, at most, complementation of the E1 and E4 regions ofthe adenoviral genome for replication.
 6. The method of claim 1 whereinthe non-subgroup C adenoviral vector lacks all or part of the E3 regionof the adenoviral genome.
 7. The method of claim 1, wherein theadenoviral vector is replication-competent.
 8. The method of claim 1,wherein the heterologous nucleic acid sequence encodes an env, gag, orpol protein from clades A, B, or C of a human immunodeficiency virus(HIV), or a fusion protein comprising any of the foregoing.
 9. Themethod of claim 1, wherein the heterologous nucleic acid sequenceencodes an E protein, an M protein, or a spike protein of a severe acuterespiratory syndrome (SARS) virus.
 10. The method of claim 1, whereinthe heterologous nucleic acid sequence encodes a transforming growthfactor β (TGFβ), an antibiotic, a malaria protein, or a nitric oxidesynthase.
 11. The method of claim 1, wherein the heterologous nucleicacid sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or3D of a foot-and-mouth disease virus (FMD).
 12. The method of claim 1,wherein the cell comprises a cellular genome into the nuclear genome ofwhich is inserted the open reading frame-6 (ORF-6) and no other openreading frame of the E4 region of an adenoviral genome operably linkedto a promoter, and which cell line complements in trans a non-subgroup Cadenoviral vector comprising an adenoviral genome having a deletion ofthe E1 and E4 regions of the adenoviral genome.
 13. The method of claim12, wherein the ORF-6 of the E4 region of the adenoviral genome isoperably linked to an inducible promoter.
 14. A system comprising: (a) acell comprising a cellular genome comprising a nucleic acid sequenceencoding a tetracycline operon repressor protein (tetR), which can beexpressed to produce tetR, and (b) a non-subgroup C adenoviral vectorhaving an adenoviral genome comprising a heterologous nucleic acidsequence, wherein (1) the heterologous nucleic acid sequence (i) encodesa protein that inhibits propagation of the non-subgroup C adenoviralvector in the cell and (ii) is operably linked to a promoter and one ormore tetracycline operon operator sequences (tetO), (2) the non-subgroupC adenoviral vector can transfect the cell and be propagated in thecell, and (3) the system lacks tetracycline.
 15. The system of claim 14,wherein the non-subgroup C adenoviral vector is replication-deficient.16. The system of claim 15, wherein the non-subgroup C adenoviral vectorrequires, at most, complementation of the E1 region of the adenoviralgenome for replication.
 17. The system of claim 15, wherein thenon-subgroup C adenoviral vector requires, at most, complementation ofthe E4 region of the adenoviral genome for replication.
 18. The systemof claim 15, wherein the non-subgroup C adenoviral vector has anadenoviral genome devoid of all of the E1 region and at least a portionof the E4 region, and the adenoviral vector requires, at most,complementation of the E1 and E4 regions of the adenoviral genome forreplication.
 19. The system of claim 14, wherein the non-subgroup Cadenoviral vector lacks all or part of the E3 region of the adenoviralgenome.
 20. The system of claim 14, wherein the non-subgroup Cadenoviral vector is replication-competent.
 21. The system claim 14,wherein the heterologous nucleic acid sequence encodes an env, gag, orpol protein from clades A, B, or C of a human immunodeficiency virus(HIV), or a fusion protein comprising any of the foregoing.
 22. Thesystem of claim 14, wherein the heterologous nucleic acid sequenceencodes an E protein, an M protein, or a spike protein of a severe acuterespiratory syndrome (SARS) virus.
 23. The system of claim 14, whereinthe heterologous nucleic acid sequence encodes a transforming growthfactor β (TGFβ), an antibiotic, a malaria protein, or a nitric oxidesynthase.
 24. The system of claim 14, wherein the heterologous nucleicacid sequence encodes protein 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, or3D of a foot-and-mouth disease virus (FMD).
 25. The system of claim 14,wherein the cell comprises a cellular genome into the nuclear genome ofwhich is inserted the open reading frame-6 (ORF-6) and no other openreading frame of the E4 region of an adenoviral genome operably linkedto a promoter, and which cell line complements in trans a non-subgroup Cadenoviral vector comprising an adenoviral genome having a deletion ofthe E1 and E4 regions of the adenoviral genome.
 26. The system of claim25, wherein the ORF-6 of the E4 region of the adenoviral genome isoperably linked to an inducible promoter.